1. Field of the Invention
The present invention relates to antibodies that bind a TSG101 protein and inhibit or reduce viral production. The invention also relates to methods of using the TSG101 antibodies for the treatment of viral infections, including HIV infection. The invention further relates to methods of detecting viral infected cells using TSG101 antibodies.
2. Background of the Technology
Pathogen and host cell interactions play critical roles in the pathogenesis of viral diseases such as AIDS. For a typical viral infection, viruses have to attach to the host cells through cell surface receptors, fuse with host cell membrane, translocate across the cell membrane, uncoat viral particles, synthesize and assemble viral proteins using host protein synthesis machinery, and release from host cells through host exporting machinery. The interplay between the viruses and host cells determine the outcome of viral pathogenesis, ranging from the elimination of viruses to a parasitic or lethal infection. For example, HIV employs a variety of strategies to productively infect human cells. A retrovirus, its life cycle begins by attaching to host cells—the primary target is the CD4+ T helper cells and gaining entry via specific receptors. In the cell, the RNA genome is “reverse” transcribed to its complementary DNA, and then shuttled to the nucleus for its integration in the host genome. This integrated “provirus” then directs the production of new viral RNA and proteins, which self-assemble and then “bud” from the cell as mature- and infectious-viral particles, enveloped in plasma membrane. Like all viruses, the HIV is a parasite, unable to catalyze the membrane fission event that drives the budding process. Instead, the nascent virus recruits the cell's membrane sorting machinery to complete this final stage of infection. Such an host and virus interplay has been well demonstrated in individuals, who carry a defective cell surface receptor (CCR5), are completely resistant to HIV infection, elucidating the important roles of host genes and genetic pathways in viral pathogenesis.
Tumor Susceptibility Gene 101 (TSG101, Li, et al., 1996, Cell 85, 319-29) plays important roles in cell growth (Zhong, et al., 1998, Cancer Res. 58, 2699-702; Oh, et al., 2002, Proc. Natl. Acad. Sci. USA 99, 5430-5; Krempler, et al., 2002, J. Biol. Chem. 277, 43216-23; Wagner, et al., 2003, Mol. Cell. Biol. 23, 150-62; Li, et al., 1996, Cell 85, 319-29), cellular protein trafficking (Babst, et al., 2000, Traffic 1, 248-58; Bishop, et al., 2002, J. Cell Biol. 157, 91-101), and degradation of p53 (Li, et al., 2001, Proc. Natl. Acad. Sci. USA 98, 1619-24; Ruland, et al., 2001, Proc. Natl. Acad. Sci. USA 98, 1859-64; Moyret-Lalle, et al., 2001, Cancer Res. 61, 486-8). TSG101 is also widely recognized as a key player in this final stage, inhibition of cellular TSG101 blocks the budding process of HIV. Acting in concert with other cellular factors, TSG 101 thus plays an essential role in the budding or spread of HIV viruses. The HIV Gag protein, previously shown to orchestrate viral assembly and budding, binds with high affinity to TSG 101, and this Gag/TSG101 interaction is essential for efficient HIV viral assembling and budding, as disruption of the Gag/TSG101 interaction prevents HIV viral budding, and significantly limit the spread of HIV virus.
The final step in the assembly of an enveloped virus assembly requires separation of budding particles from the cellular membranes. Three distinct functional domains in Gag, 20, i.e., PTAP in HIV-1 [SEQ ID NO.: 44] (Gottlinger, et al., 1991, Proc. Natl. Acad. Sci. USA 88, 3195-9; Huang, et al., 1995, J. Virol. 69, 6810-8); PPPY in RSV [SEQ ID NO.: 45] (Parent, et al., 1995, J. Virol. 69, 5455-60), MuLV (Yuan, et al., 1999, Embo. J. 18, 4700-10), and M-PMV (Yasuda, et al., 1998, J. Virol. 72, 4095-103); and YXXL in EIAV (Puffer, et al., 1997, J. Virol. 71, 6541-6), have been identified in different retroviruses that are required for this function and have been termed late, or L domains (Wills, et al., 1991, Aids 5, 639-54). In HIV-1, the L domain contains a PTAP motif and is required for efficient HIV-1 release (see, e.g., Wills, et al., 1994, J. Virol. 68, 6605-6618; Gottlinger, et al., 1991, Proc. Natl. Acad. Sci. USA 88, 3195-3199; Huang, et al., 1995, J. Virol. 69, 6810-6818). The L domain of HIV-1 p6, especially the PTAP motif, binds to the cellular protein TSG101 and recruits it to the site of virus assembly to promote virus budding (VerPlank, et al., 2001, Proc. Natl. Acad. Sci. USA, 98:7724-7729; Garrus, et al., 2001, Cell 107:55-65; Martin-Serrano, et al., 2001, Nature Medicine 7:1313-19; Pornillos, et al., 2002, EMBO J. 21:2397-2406; Demirov, et al., 2002, Proc. Natl. Acad. Sci. USA 99:955-960; PCT Publication WO 02/072790; U.S. Patent Application Publication No. US 2002/0177207). The UEV domain of TSG101 binds the PTAP motif and mono-ubiquitin (Pornillos, et al., 2002, Embo J. 21, 2397-406; Pornillos, et al., 2002, Nat. Struct. Biol. 9, 812-7), which has also been implicated in HIV-1 budding (Patnaik, et al., 2000, Proc. Natl. Acad. Sci. USA 97, 13069-74; Schubert, et al., 2000, Proc. Natl. Acad. Sci. USA 97, 13057-62; Strack, et al., 2000, Proc. Natl. Acad. Sci. USA 97, 13063-8). Depletion of cellular TSG101 (Garrus, et al., 2001, Cell 107:55-65) or over-expression of a truncated form of TSG101 inhibits HIV-1 release (Demirov, et al., 2002, Proc. Natl. Acad. Sci. USA 99:955-960). Under certain circumstances, TSG101 can even substitute for the HIV-1 L domain to promote virus release (Martin-Serrano, et. al., 2001, Nature Medicine 7:1313-19).
In yeast, the Tsg101 ortholog Vps23 has been shown to interact with Vps28 and Vps37 and to form a protein complex named ESCRT-I, which is critical for endosomal protein sorting into the multivesicular body pathway (Katzmann, et al., 2001, Cell 106, 145-55). It is hypothesized that this intracellular multivesicular body formation resembles HIV-1 release at the plasma membrane (Garrus, et al., 2001, Cell 107:55-65; Patnaik, et al., 2000, Proc. Natl. Acad. Sci. USA 97, 13069-74). In mammalian cells, TSG101 interacts with Vps28 to form an ESCRT-I-like complex (Babst, et al., 2000, Traffic 1, 248-58; Bishop, et al., 2002, J. Cell Biol. 157, 91-101; Bishop, et al., 2001, J. Biol. Chem. 276, 11735-42), although the mammalian homolog of Vps37 has not been identified.
Recent studies (Blower, et al., 2003, AIDS Rev. 5:113-25; Valdiserri, et al., 2003, Nat. Med. 9:881-6) have estimated that as many as 42 million people worldwide have been infected with HIV. The disease has killed more than 3 million people. While the advent of highly potent and targeted combination therapies has slowed the progression of AIDS in industrialized nations, the AIDS pandemic is causing a “human development catastrophe” in developing nations, particularly in Africa, where more than 21 million Africans have been infected. In South Africa alone, the death toll is projected to rise to 10 million by 2015. Related statistics portend a similar crisis in the Asia Pacific region, which, according to United Nations' estimates, has more than 7 million HIV-infected individuals. Repercussions from the AIDS pandemic extend well beyond the clinic, which lack the resources to treat the swelling numbers of recently infected patients (nearly 20% of the adult population in South Africa is infected). Treatment of HIV-infected and gravely ill AIDS patients is stressing the already over-burdened health care systems of Africa and other developing nations. Worse yet, current treatments for HIV □ despite their initial success in reducing viral load □ are beginning to lose their efficacy, as drug-resistant HIV strains are increasingly isolated in newly infected individuals. Further compounding the therapeutic management of HIV disease is the toxicity of current antiretroviral regimens, the magnitude of which complicates the physician's decision to begin and to maintain treatment. Identifying new therapeutic paradigms for the treatment of HIV disease, especially those with mechanisms of action that promise to slow the development of resistance, is indeed a global challenge for the pharmaceutical industry.
Many viruses are also highly mutable. Methods and compositions relying on targeting such viruses directly are normally not sufficient in the treatment of infection by such viruses. For example, HIV-1 is such a highly mutable virus that during the course of HIV-1 infection, the antibodies generated in an infected individual do not provide permanent protective effect due in part to the rapid emergence of neutralization escape variants (Thali, et al., 1992, J. Acquired Immune Deficiency Syndromes 5:591-599). Current therapies for the treatment of HIV-infected individuals focus primarily on viral enzymes involved in two distinct stages of HIV infection, the replication of the viral genome and the maturation of viral proteins. Since the virus frequently mutates, strains resistant to an antiviral inhibitor develop quickly, despite the drug's initial therapeutic effects. In one recent study, the percentage of individuals newly infected with drug-resistant HIV strains increased six fold over a five year period (Little, et al., 2002, N. Engl. J. Med. 347:385-94). Further, combination therapy, the current standard of care that attacks HIV with inhibitors of both reverse transcriptase and protease, is leading to the development of multi-drug resistant HIV strains. Anti-retroviral drugs directed against new HIV-based targets, while of considerable value, do not address this increasingly critical issue. For example, HIV strains resistant to Fuzeon® (enfuvirtide), the newest addition to the anti-HIV armamentarium, have already been isolated from patients. Thus, despite its antiviral potency and novel mechanism of action, drug-resistance is likely to undermine the therapeutic potential of viral fusion inhibitors like Fuzeon®. There is therefore a need for developing novel therapeutics and preventative measures to combat viral infections such as HIV infection.
Discussion or citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.